We have recently shown that invariant chain (Ii) has dramatic effects on the biosynthesis of Class II. Class II that is expressed in the absence of Ii, misfolds in the ER losing some monoclonal antibody epitopes, is inefficiently transported to the Golgi, is more heavily glycosylated within the Golgi, and at the cell surface more efficiently presents peptide antigens to T cells. Surprisingly, Ii-positive cells did not process and present antigen better than Ii-negative cells, although preliminary data suggest that the alternatively spliced form of Ii, p4l, may be critical for this effect. In addition, we have found that the chondroitin sulfate form of Ii (Ii-CS) is necessary for stimulation of primary allogeneic and mitogenic T cell responses. The observations that Ii-CS can be isolated from the plasma membrane and that the T cell marker CD44 binds to chondroitin sulfate suggest that the Ii-CS may function as an adhesion molecule at the cell surface. The overall goal of this proposal is to explore the effects of Ii on Class II biosynthesis, antigen presentation, and T cell activation. Specifically, we will examine the effects of Ii on the post-Golgi transport of Class II and the relative importance of the various forms of Ii on the ability of Class II to associate with processed exogenous antigens within this post-Golgi compartment. In addition, it has been suggested that Ii may function by occupying the Class II antigen binding site early in biosynthesis, effectively inhibiting Class II from associating with peptides within the ER. Ii dissociation in an endosomal compartment would allow for association of Class II with exogenous antigenic peptides. We will test this model directly by comparing the ability of Ii-positive and Ii-negative cells to present endogenous antigen to Class II restricted T cells. To address the function of the Ii proteoglycan we will determine whether Ii-CS functions as an accessory molecule at the cell surface of the APC or whether Ii-CS modifies the conformation or biosynthesis of the majority of or a subset of Class II molecules. Finally, we will generate a mutant mouse line that do not express Ii by gene targeting in embryonic stem cells. These mice will enable us to determine if Ii influences the development of the T cell repertoire in the thymus as well as effecting peripheral antigen presenting cells.